Rotaviruses are an important cause of viral gastroenteritis in humans and animals. The rotavirus genome consists of eleven monocistronic segments of double-stranded (ds)RNA which encode six structural (VP1, VP2, VP3, VP4, VP6 and VP7) and five nonstructural proteins (NSP1, NSP2, NSP3, NSP4, NSP5). The plus-strand of the genome segments is indistinguishable from viral mRNA, containing a 5'-terminal methylated cap structure but lacking a 3'-poly(A) tail. Except for short sequences of seven to ten nucleotides at the 5'- and 3'-termini, the eleven genome segments share no detectable homology. The conserved terminal sequences have been shown to contain cis-acting signals that are important in genome replication. In contrast, the role of the conserved termini and the 5'- and 3'-noncoding sequences (NCS) in regulating rotavirus gene expression is not known. Previous studies have established that while the eleven genome segments exist in equimolar concentrations in the infected cell, transcription of the segments leads to the synthesis of non-equimolar levels of viral mRNA. The relative levels of the eleven viral proteins made in the infected cell do not reflect the relative levels of viral mRNA that are present indicating that cis-acting signals in the viral mRNAs are important for regulating rotavirus gene expression. The goal of this project is to identify those cis-acting signals in the 5'- and 3'-NCS that affect expression of the viral proteins. Initial studies examined the contribution of the 5'- and 3'-NCS on translation using synthetic analogs of rotavirus mRNAs. These analogs consisted of the gene for bacterial chloramphenicol acetyltransferase (CAT) flanked by the 5'- and 3'-NCS of the rotavirus genes 1, 2, 3, 4, 6 or 9 and were assayed for translational efficiency in rabbit reticulocytes. The results revealed: (i) analogs carrying homologous 5'- and 3'-NCS of gene 6 induced the highest levels of CAT expression, (ii) the 3'-NCS of RNA segment 6 could enhance the translation efficiency of other rotavirus mRNA analogs regardless of the 5'-NCS, (iii) the translation enhancement element was localized to a region approximately 80 nucleotides upstream from the 3'-end of the 3'-NCS of RNA segment 6. Our recent studies have compared the translational efficiency of wildtype gene 6 mRNA with gene 6 mRNA containing deletions in the 3'-NCS. The results confirmed the results obtained with the rotavirus-CAT analogs and establish that an important cis-acting element resides in the 3'-NCS of rotavirus gene 6 mRNA which functions to up regulate the expression of the gene 6 product, VP6, a major protein component of the rotavirus virion.